final report rev 07...2 List of Tables. Table number Table Name Page 5-1 Presence vs. Stream order...

Mannonphryne olmonae:

An Ecological Assessment

in Tobago,

Republic of Trinidad and Tobago Project Number: 300206

Project L.E.A.P.:

Jahson B. Alemu I, Michelle N. E. Cazabon, Lean

Dempewolf,

Ryan P. Mannette, Kerrie T. Naranjit and

Alicia Schimdt-Roach.

Assisted by Dr. Adrian Hailey

© J. Alemu I, M. Cazabon, L. Dempewolf, R. Mannette, K. Naranjit and A. Roach 2007.

Not to be reproduced without permission from Project L.E.A.P.

Contact: Jahson Alemu I: [email protected]

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Table of Contents

1 Introduction................................................................................................................. 2 1.1 Background ......................................................................................................... 3 1.2 Species description.............................................................................................. 4 1.3 Habitat description .............................................................................................. 4 1.4 Batrachochytrium dendrobatidis......................................................................... 5

2 Aims and Objectives ................................................................................................... 6 2.1 General Aim........................................................................................................ 6 2.2 Objectives: .......................................................................................................... 6

3 Site Description........................................................................................................... 7 4 Field Method............................................................................................................. 10

4.1 Distribution, Presence and Abundance ............................................................. 10 4.2 Ecology and Habitat Assessment...................................................................... 10 4.3 Chytrid Survey .................................................................................................. 11 4.4 Statistical Analysis............................................................................................ 12

5 Results....................................................................................................................... 13 5.1 Presence Survey ................................................................................................ 13 5.2 Population Structure.......................................................................................... 18 5.3 Habitat Assessment ........................................................................................... 20

5.3.1 Tadpole Microhabitat................................................................................ 20 5.3.2 Adult Microhabitat.................................................................................... 21

5.4 Batrachochytridium dendrobatidis d Survey .................................................... 24 6 Discussion ................................................................................................................. 26

6.1 Habitat, Abundance and Population dynamics ................................................. 26 6.2 Batrachochytrium dendrobatidis....................................................................... 27

7 Measures for Conservation ....................................................................................... 29 7.1 Future of Mannophryne olmonae...................................................................... 29 7.2 Education and Public Awareness programme .................................................. 30 7.3 Follow up Activities.......................................................................................... 30

8 References................................................................................................................. 33

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List of Tables. Table number Table Name Page 5-1 Presence vs. Stream order 13 5-2 Presence vs Watershed 15 5-3 Stream Order 1 Comparison 16 5-4 Comparison of watersheds using second and third

orders streams only 16

5-5 Frog presence vs. Presence of tributaries 17 5-6 Predators vs. Frog Presence 17 5-7 M. olmonae abundance along survey rivers 18 5-8 Physical characteristics of pools with and without M.

olmonae tadpoles 20

5-9 Measured Environmental Variables 21 5-10 PCR Positives for B. dendrobatidis 24 List of Figures Figure Figure Name Page 3-1 Study Rivers 8 3-2 Watersheds system of the Main ridge Forest Reserve 9 5-1 Surveyed Areas 14 5-2 Size frequency distributions (numbers observed) of

Mannophryne olmonae: a) juveniles and females (n = 110); b) males (n = 16).

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5-3 Frequency distributions (numbers observed) of: distances of M. olmonae frogs from the river or stream edge (n = 68)

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5-4 Areas screened for B. dendrobatidis 24 List of Appendices Appendix A: Tadpole Key Appendix B: Batrachochytrium dendrobatidis sampling Appendix C: Raw Data Appendix D: PCR Results

3

1 Introduction

1.1 Background

The Bloody Bay Poison Frog, Mannophryne olmonae is endemic to the forests of the

Main Ridge Forest Reserve on the island of Tobago, of the Republic of Trinidad and

Tobago, and listed as Critically Endangered (Baillie, 2004; IUCN, 2004). Hardy (2004)

estimated an 80% population decline over the last three generations, with the population

limited to an area less than 100 km2. Hardy’s assessment also indicated that M.

olmonae’s had limited geographic range (restricted to north-east Tobago), a severely

fragmented distribution, with continuing decline in the number of mature individuals may

have also contributed to this decline. All these factors contributed to its designation as

critically endangered.

From a global perspective, this decline is another example of the phenomenon affecting

amphibian populations worldwide, potentially leading to amphibian species extinction.

The major threats to amphibian populations include habitat loss and urbanisation, disease,

global warming, pollution, unsustainable use, predation and unknown declines (Gibbons

2000).

All species of the genus Mannophryne are found in Venezuela and the island state of

Trinidad and Tobago. Most of the geographical range is dominated by montane areas,

although some species reach down to sea level. Members of this genus inhabit humid

forests with a mean annual precipitation of 1100 to 2200mm, and mean annual

temperatures of 18 to 24°C, parameters that may constitute useful predictors of the

occurrence of these frogs in the northern mountains of South America (La Marca 1992).

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1.2 Species description

The Bloody Bay frog Mannophryne olmonae was described by Hardy (1983), who

separated the populations on Tobago from M. trinitatis of Trinidad and Venezuela with

which it was formerly included. Subsequently the Venezuelan populations of these

aromobatid (formerly dendrobatid – (Grant 2006)) stream frogs have also been separated

into a new species (Barrio-Amorós et al., 2006; M. J. Jowers, pers. comm.), leaving

endemic Mannophryne species in Tobago.

Mannophryne olmonae belongs to the family Arombatidae, but unlike many close

relatives it is non-poisonous and dull coloured. This species can be distinguished from

the other locally found brown frogs, M. trinitatis, by a narrow collar which is uniformly

coloured and by its call (La Marca 1992). The genus is diurnal and males typically call

throughout the day. Males may reach 25mm and females 28mm in length, (Hardy 1983).

Sexes are distinguished by the bright yellow chin and black collar in females and

grey/black chin in males, however, during the breeding season, males turn jet black while

calling.

The eggs are laid on land; hatching tadpoles (11 to 19 in number) are carried to streams

by the male where they complete their development (IUCN, 2004). Almost nothing is

known about natural prey items of Mannophryne; unpublished data indicate that some

Mannophryne species feed on beetles, large ants and mites (La Marca 1992).

1.3 Habitat description

The Bloody Bay Frog is a tropical forest species usually found in upland areas of north-

eastern Tobago at elevations above 120m along streams (typically clear-water rivers, of

fast velocity in the upper streams, with substrates of stones, gravel and silt and banks of

boulders and gravel ) between the river edge and forest edge (Hardy, 2004). Although,

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the geographical range is dominated by mountains, M. olmonae has been noted to reach

down to sea level (La Marca 1992).

1.4 Batrachochytrium dendrobatidis

The chytrid fungus Batrachochytrium dendrobatidis (B. dendrobatidis) is the pathogen

responsible for the amphibian disease chytridiomycosis (a disease of the keratinised

skin). It is thought to be one of the main causes of the global decline in frog populations

since the 1960s, and the dramatic population crashes from the 1970s onwards (Parris and

Beaudoin, 2004). In areas such as Australia and Central America, chytridiomycosis is

thought to be causing the decline of anurans (Berger, et al. 1998; Lips 1999); and may

also be responsible for the loss of otherwise healthy animals (Daszak and Cunningham

1999). Some amphibian biologists consider B. dendrobatidis as a major cause of

population declines of amphibian species confined to most montane rain forests

worldwide (Daszak 1999; Weldon et al. 2004). B. dendrobatidis has been found in most

amphibian populations on every continent, including Asia: Africa, Australasia-Pacific,

North America, and South America (Lips et al. 2003; Weldon et al. 2004). To date, there

has been limited assessment of the spread of the pathogen thoroughly in the Caribbean,

but has been detected in countries such as Puerto Rico (Burrowes 2004) and Dominica

(Fa 2004)). Worldwide, extensive research is being done to track the spread and attempts

have been made to curb the spread of the pathogen.

Amphibian species likely to decline from B. dendrobatidis are stream-associated

(McDonald and Alford 1999), endemic, have a large body size and occur at high

elevations (Lips, Reeve et al. 2003). This pathogen is of particular concern to Tobago, a

relatively small island (300 km²) with a high potential to spread to the other 14 species of

amphibians on the island.

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2 Aims and Objectives

2.1 General Aim

To determine the ecological status of M. olmonae, while assessing possible threats facing

the species.

2.2 Objectives:

1. To assess the population status (numbers and range), ecological needs, and

microhabitat and habitat status using rapid assessment protocols.

2. To develop a viable GIS database allowing the continued study of M. olmonae

and other amphibians.

3. To determine the presence of the chytrid fungus and the extent of its spread.

4. To promote long term conservation of local endangered species (and habitats) and

biodiversity in and around the Main Ridge Forest Reserve. The use of educational

and training workshops, geared toward producing conservation leaders/managers

will provide a sustainable pillar for further research and development in the

environmental sector.

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3 Site Description

The Main Ridge extends from the Northeast tip of the island Southwest for about two

thirds of the length of Tobago, achieving a maximum altitude of 580m. The forested

ridge rises steeply from the North coast and the gentler southern slopes are deeply

indented by valleys which run down to a narrow but fertile coastal plain. The tropical

rain forest is restricted to the sheltered mountain valleys of the Main Ridge. Lower

montane forest, xerophytic rain forest, evergreen formations and some elfin woodland

also occurs (Davis et al. 1986; Thelen and Faizool 1980). The rainfall regime is seasonal,

with less during the dry season (January to May) and more during the wet season (June to

December).

Six major rivers with headwaters in the Main Ridge Forest Reserve, as well as several

short streams along the Northeast coast of Tobago were identified and assessed for the

presence and density of M. olmonae. The criteria for which rivers were selected were

based on the type localities of the frog established by Murphy (1997). The study rivers

selected along Northside Road were not listed as type localities but were discovered to

have M. olmonae present

Sample rivers included: Doctor’s River, Louis D’or River, Argyle River, Roxborough

River, Kings Bay River, Bloody Bay River, and several small rivers along the Northside

Road (see Figures 3.1 and 3-2).

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Figure 3.1: Study Rivers

9

Figure 3-2: Watersheds system of the Main ridge Forest Reserve

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4 Field Method

4.1 Distribution, Presence and Abundance

The local distribution of M. olmonae along each river was determined using visual and

aural encounter methods (walking the length of the river). The distances from the mouth

of the river at which any M. olmonae were observed along the respective watercourses

were noted along with the distance along the river walked. Rock pools were also

examined for M. olmonae tadpoles. Tadpoles were compared against an identification

key adapted from Kenny (1983), (see Appendix A). When calling was heard, the

presence and the number of discernable frogs heard were noted. In conjunction with

presence, an abundance survey was conducted (adults and tadpoles) along the main river

and its tributaries. Further, the calling range was estimated by moving away from a

calling male, until it was no longer heard, and then moving back toward the point that it

was heard again. This distance was noted as the audibly detectible calling radius.

4.2 Ecology and Habitat Assessment

The narrowed study area surveyed included a further 250m on either end of the range of

detected presence. Where adult M. olmonae were encountered, the following data was

collected: activity before disturbance, distance along river, distance from the river,

distance from the forest edge, sex, presence of tadpoles on the male’s back, deformities,

injuries or parasites and snout-vent length (SVL) to the nearest 0.1mm.

Where frog choruses were heard, the estimated number of frogs calling was recorded as

well as the distance along the river that the chorus was heard.

1. If M. olmonae adults were encountered between quadrats, they were

omitted from the density survey, but were recorded as part of the presence

survey.

2. Frogs were released at the place of capture after examination

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Where tadpoles were encountered the following data was collected: the number and size

M. olmonae tadpoles, the approximate area and depth of the isolated pools (to the nearest

0.1 cm), the temperature, pH and Dissolved Oxygen content in the pools, the distance of

the pool from the main river, GPS coordinates of isolated pools, the type of the

substratum at the bottom of the pools, the relative abundance of other macrofauna

(mosquito larva, crayfish etc) in the pools, the state of development of tadpoles (e.g. with

or without legs).

Habitat data collected included: the percentage canopy cover, the river velocity, the

substrate type, the dominant vegetation, the width of river, and bank, and the physico-

chemical parameters in the river (temperature, pH and dissolved oxygen level).

All other frog species seen or heard during the survey were recorded, but no quantitative

data were collected. This data was also used to develop and update species notes on the

species (to be published)

4.3 Chytrid Survey

Skin swabs were collected from some of the frogs caught, as described below, for

obtaining Batrachochytrium dendrobatidis (B. dendrobatidis) samples for PCR testing.

Sampling was conducted during the months May to August using the protocol outlined by

Lauren J. Livo (Methods for obtaining Batrachochytrium dendrobatidis (BD) samples for

PCR testing) (see Appendix B). Each frog was handled with a new pair of disposable

latex gloves covering the hands in order to avoid cross-contamination between

individuals. The body surfaces, especially the posterior ventral surface of the frogs were

swabbed 25 times. Swabs were then stored in individual tubes with 75% ethanol, and

kept out of direct contact with any nucleotide denaturing agents. The diagnostic PCR

tests were performed using the Taqman RT-PCR by Pisces Molecular (5311 Western

Avenue, Suite E, Boulder, CO 8030).

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4.4 Statistical Analysis

All statistical permutations were done using the statistical software MINITAB Release

13.32 for Windows. Binary logistic regressions were conducted to examine the

relationship between the measured habitat parameters which were assumed to potentially

affect the presence of M. olmonae and the presence/absence data (binary response). All

means are followed by ± SE. Alpha was set at 0.05.

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5 Results

5.1 Presence Survey

M. olmonae was detected along all rivers over a total distance of 22.05 km. Figure 5-1

shows the general upland reaches within which they were detected.

a) Stream Order

Chi squared association test was conducted to determine the relationship between stream

order and frog presence.

Table 5-1: Presence vs Stream order

Stream Order Frogs Quadrats Total % Presence

Absent Present

1 17 16 33 48

2 86 10 96 10

3 250 39 289 13

4 23 0 23 0

Combining stream orders 3 & 4 for analysis due to small sample of 4th order streams,

chi-sq = 32.58, 2 df, P < 0.001.

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Figure 5-1: Surveyed Areas

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b) Watersheds

Further comparison of presence was conducted within watersheds of the Main Riodge

Forest Reserve (MRFR). North coast tributaries were considered as one watershed.

Table 5-2: Presence vs Watershed

Watershed Frog Quadrats Total % Presence

Absent Present

1(DR) 24 22 46 48

2(LD) 142 21 163 13

3(BB) 88 5 93 5

4(RR) 80 2 82 2

5(KB) 27 3 30 10

6(AR) 6 2 8 25

7(NCT) 9 10 19 53

1: Doctor’s River; 2: Louis D’Or River; 3: bloody Bay River; 4: Roxborough River; 5: king’s Bay River; 6:

Argyle River; 7: North Coast Rivers

Excluding Argyle River as sample too small, chi-sq = 80.0, 5 df, P < 0.001.

c) Interaction between watershed and stream order.

This is to see if stream order explains variation among watersheds or whether there was

significant variation after taking differences in stream orders into account. Doing a 3-way

analysis results in too small samples, so this was done in two stages:

First stage, compare first order streams which were part of rivers (only Doctor’s River &

Roxborough River had first order streams sampled), with the North Coast streams which

were not part of larger rivers:

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Table 5-3: Stream order 1 comparison

Stream type Frogs Quadrats Total % Presence

Absent Present

Stream order 1

(river)

8 6 14 43

Stream order 1

(Stream)

9 10 19 53

Chi-sq = 0.31, 1df, P = 0.579. Therefore, first order streams had high presence of frogs,

whether they were isolated streams or tributaries of rivers.

Second stage, compare the watersheds using only second and third order streams, which

had similar overall levels of 10-13% presence, but exclude fourth order streams which

had no presence (analysis a) above):

Table 5-4: Comparison of watersheds using second and third orders streams only

Watershed Frog Quadrats Total % Presence

Absent Present

1 DR 2+3 22 17 39 44

2 LD 2+3 142 21 163 13

3 BB 2+3 68 5 73 7

4 RR 2+3 73 1 74 1

5 KB 2+3 25 3 28 11

6 AR 2+3 6 2 8 25

Excluding Argyle River as sample too small, chi-sq = 45.2, 4 df, P < 0.001

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d) Presence of tributaries.

Was presence of frogs associated with presence of a tributary in higher order streams?

Table 5-5: Frog presence vs. Presence of tributaries

Stream Order

2-4

Frogs Quadrats Total % Presence

Absent Present

No tributary1 336 43 379 11

Tributary

present

23 6 29 21

Chi-sq = 2.23, 1df, P = 0.136

e) Predators

Did the presence of predators affect the presence of these stream frogs.?:

Table 5-6: Predators vs. Frog Presence

Stream Order Frogs Quadrats Total % Presence

Absent Present

Abesnt 32 13 45 29

Present 59 35 94 37

Chi-sq = 0.94, 1 df, P=0.333.

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5.2 Population Structure

Table 5-7 below shows the abundance of frogs found within the surveyed areas.

Table 5-7: M. olmonae abundance along survey rivers.

River Survey Area (m2) Abundance

Doctor’s River 2536.5 125

Louis D’Or river 20586.9 28

Bloody Bay river 2240.0 8

Roxborough River 1312.5 2

Argyle River 3000.0 50

King’s bay River 820.0 1

T2 280.0 1

T4 610.0 1

T5 65.0 7

T18 170.0 8

T20 2536.5 9

A total of 126 M. olmonae were measured (Fig.5-2),. The smallest juvenile had a SVL of

9.6 mm. Male M. olmonae ranged from 18.3-25.7 mm. Females also had a maximum

size of 25.7 mm

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Figure 5-2. Size frequency distributions (numbers observed) of Mannophryne

olmonae: a) juveniles and females (n = 110); b) males (n = 16)

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5.3 Habitat Assessment

5.3.1 Tadpole Microhabitat

Eighteen small rock pools close to the river were inspected, nine of these contained M.

olmonae tadpoles. All M. olmonae tadpoles were found in rock crevices adjacent to the

river or stream with the exception of one locality, a first-order stream near Bloody Bay,

where they were found in water-filled tyre ruts. Despite the fact that all of the collection

localities of tadpoles were directly adjacent to streams, no M. olmonae tadpoles were seen

in the streams themselves. None of the physical characteristics measured differed

significantly between pools with and without M. olmonae tadpoles.

Tadpole pools were identified at three locations: Doctor’s River, Bloody Bay River and

Argyle River. The dimensions and physico-chemical characteristics of these pools are

summarised in Table 5-8 below.

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Table 5-8: Physical characteristics of pools with and without M. olmonae tadpoles.

Values are mean ± SD (with number of pools), and range for pools with tadpoles,

and the P value from an unequal-variance t test comparing the means.

Pool

Parameter Overall

Pools with M.

olmonae

Pools without

M. olmonae P

Length (m) 73 ± 69 (8) 20-200 58 ± 35 (9) 0.586

Width (m) 43 ± 33 (8) 11-100 19 ± 8 (9) 0.085

Depth (m) 9.9 ± 7.2 (8) 3.0-25.0 8.0 ± 6.6 (9) 0.581

pH 7.7 ± 0.7 (7) 6.2-8.0 8.1 ± 0.3 (8) 0.156

Temperature

(ºC) 27.4 ± 1.3 (7) 25.2-28.6 27.2 ± 1.0 (8) 0.751

DO (mg L–1) 3.2 ± 1.8 (7) 1.72-6.95 3.3 ± 3.0 (8) 0.942

Distance from

River (m) 2.9 ± 1.3 (6) 1.2-5.1 1.7 ± 1.2 (8) 0.113

Distance from

Forest (m) 1.9 ± 1.4 (6) 0-4.0 1.7 ± 1.9 (8) 0.831

5.3.2 Adult Microhabitat

In order to assess the effect of different habitat variables on the presence of M. olmonae

regression analyses were conducted on those environmental factors. The environmental

variables were selected on the assumption that they had the greatest potential of affecting

the presence and number of M. olmonae. Table 5-9 below shows the mean of measured

environmental variables in which mature frogs were found across all the rivers.

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Table 5-9: Measured Environmental Variables

Environmental variables Frog Present Frog Absent

River Width (m) 2.09 ± 3.02(65) 2.94 ± 3.33 (119)

River Depth (m) 0.21 ± 0.18 (61) 0.25 ± 0.25 (117)

Velocity (m/sec) 0.32 ± 0.19 (58) 0.35 ± 0.19 (116)

% Canopy Cover 55.45 45.67 ± (118)

pH 8.25 ± 0.87 (42) 8.19 ± 0.92 (77)

Temperature (°C) 27.03 ± 1.09 (60) 26.52 ± 2.41 (119)

Dissolved Oxygen (mg L–1) 6.97 ± 0.65 (60) 7.19 ± 0.50 (119)

Bank Width 3.0 ± 3.64 (51) 3.29 ± 5.29 (87)

Distance to Forest Edge 3.74 ± 2.91 (63) 4.30 ± 4.30 (116)

Vegetation dominance Heliconia balisier, Heliconia balisier,

Bambusa vulgaris

Main substrate description stones, gravel and silt and

banks of boulders and

gravel

Gravel and silt with

large boulders

Main associated fauna Crayfish and fish Mountain Mullet,

crayfish

A binary logistic regression was conducted to determine the significance of the

synergistic effects of the different parameters on the presence of M. olmonae.

Inconsistencies in data collection demanded the occlusion of pH and bank width data,

from the analysis (see Appendix C). Major data gaps in these parameters would have

meant the inclusion of which would have significantly skewed the outcome of the

regression analysis. The analysis showed that temperature (Z= 2.37, P = 0.018) and

dissolved oxygen (Z= - 2.03, P = 0.043) were the only significant factors. When

temperature and dissolved oxygen were removed from the analysis, stream velocity was

the only significant factor (Z= - 2.08, P = 0.038). When dissolved oxygen, temperature

and stream velocity were removed from the model, all other factors did not significantly

affect the presence of frogs.

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In order to confirm findings of the binary logistic regression, single regressions were

conducted on the significant factors. The analysis showed that dissolved oxygen (T = -

2.54, P = 0.012) was the only factor that significantly affected the presence of M.

olmonae at these streams.

All M. olmonae that were located individually were within 10 m of the edge of the river

or stream (Figure 5-3). However calling males were occasionally heard farther away

from the water. The mean distance from the river or stream was 2.0 ± 2.6 m (n = 68), and

more than half of the individuals were within 1.0 m of the water’s edge. The mean

distance from the edge of the forest was 1.5 ± 1.1 m (n = 26), with a range from 0-5 m.

No M. olmonae were observed within the forest.

Figure 5-3: Frequency distributions (numbers observed) of: distances of M. olmonae

frogs from the river or stream edge (n = 68)

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5.4 Batrachochytridium dendrobatidis d Survey

A total of 124 skin swabs were collected for analysis, of which 91 were from M. olmonae

and 40 from other species1 (see Appendix D). Of these, a total of 21 PCR positive

samples for B. dendrobatidis were detected, and they were all detected on M. olmonae.

Overall, three sites were found to be positive for B. dendrobatidis. Table 5-10 below

summarises the results of the chytrid survey.

Figure 5-4: Areas screened for B. dendrobatidis

1 Other species included Bufo marinus and Leptodactylus sp.

25

Table 5-11: PCR Positives for B. dendrobatidis

Species Location Number of

positive Swabs

Total

number of

swabs

taken

Percentage of

swabs that

were positive

Weak = 1

Positive = 4

Strong = 9

Very strong = 5

Doctor’s River

Total = 19

72

26.4

North Coast

Road

Total = 1 (strong) 9 11.1

Argyle River Total = 1 (strong) 5 20.0

Louis D’or Total = 0 3 0.0

M. olmonae

Bloody Bay Total = 0 2 0.0

Man O War Bay Total = 0 26 0.0 Bufo marinus

Windward Rd Total = 0 6 0.0

26

6 Discussion

6.1 Habitat, Abundance and Population dynamics

Mannophryne olmonae were recorded in all surveyed areas, suggesting that this species is

still widely distributed in north eastern Tobago. The greatest abundance was noted in the

upper areas of the survey streams, where frogs were significantly more associated with

first order streams (P < 0.001) rather then higher order streams. When first order streams

which are part of rivers (Doctor’s River and Roxborough River), were compared with

North Coast streams (not part of larger rivers), there was no significant difference in frog

presence between the two, as such this further affirmed that first order streams had high

presence of frogs, regardless if there were isolated streams or tributaries of rivers.

Within the wider area, watersheds were compared using only second and third order

streams. There was also significant variation in frog presence between watersheds, even

when only considering second and first order streams. Overall, both stream order and

watershed independently had significant effects. First order streams had highest presence

of frogs, whether they were isolated streams or parts of rivers. Second and third order

streams had lower presence of frogs, and this varied significantly among watersheds.

The highest abundance was noted within the Doctor’s River watershed and the north

coast watersheds. The characteristic of these areas are shown in table 5-9. These streams

are generally rocky, heavily shaded narrow and relatively undisturbed by human

activities or encroachments. However, lower stream areas, where higher order streams

are found exhibited signs of habitat degradation through sand mining, agriculture, river

dredging, dumping of garbage and eco-tourism.

A total of 223 frogs were encountered during this survey, and 126 were measured for a

population structure assessment. The majority of M. olmonae captured were immature

juveniles. The dominance of females in the sample reflected the difficult with which it

was to capture males due to camouflage and location. Generally, males tended to be

farther from the water, where their black camouflage allowed them to seamlessly blend

27

into the rocky forest background. Furthermore, the adult sex ratio of approximately 2:1

did not vary significantly at the different rivers. There was no evident sexual sexual

dimorphism, where mean adult sizes were not significantly different: females 21.6 ± 2.2

mm (n = 33); males 21.0 ± 2.4 mm (n = 15) (t = 0.82, 24 df, P = 0.42).

6.2 Batrachochytrium dendrobatidis

These results demonstrate conclusively that Batrachochytrium dendrobatidis occurs in

Tobago, and in the Critically Endangered frog Mannophryne olmonae; a worrying

finding on both counts. Over 90% of the frogs surveyed were M. olmonae, with the other

species consisting of Bufo marinus, and Leptodactylus sp. Overall, 16.9 % of the samples

were PCR positive for B. dendrobatidis endrobatidis. However, infected frogs showed

neither clinical signs nor symptoms of infection (lethargy, skin lesions) nor did they

behave abnormally, which suggests B. dendrobatidis may have no effect on this species.

The lack of these clinical signs, suggest that chytridiomycosis may not be pathogenic and

responsible for the recent population declines.

One school of though have suggested that B. dendrobatidis may not be pathogenic to

some species of infected frogs due to host-pathogen co-evolution or the production of

antimicrobial skin peptides (which some studies have linked to chytridiomycosis

resistance). It is known, however, that amphibian species vary in their susceptibility to

the disease (Blaustein et al., 2005; Garcia et al., 2006). Additionally, even susceptible

species can recover to leave B. dendrobatidis isolated to this species rather than an

epidemic disease (McDonald et al., 2005; Woodhams and Alford, 2005; Kriger and Hero,

2006; Puschendorf, Bolanos et al., 2006).

Infections were not evenly distributed across all the sampled water courses, where it was

detected at three of the survey areas (see Figure 5-4). The highest incidences of

infections were detected at Doctor’s River. The vast majority of infections were limited

to the south sloping side of the Main Ridge Forest reserve (MRFR). This physical barrier

may retard the spread of the disease to the north sloping side of the MRFR.

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There are no historical population data on M. olmonae, so it is therefore unknown

whether current populations have recovered to previous levels, or remain at a reduced

density. It is also unclear whether any population decline was caused by epidemic

chytridiomycosis. Hardy’s 2004 assessment of M. olmonae in the IUCN Red List

reported an estimated 80% population decrease in recent years however the basis for this

is unclear. It is clear, however, that widely spread populations of M. olmonae currently

harbour endemic chytridiomycosis, apparently without clinical signs. This finding raises

several questions and implications for amphibian health in Tobago, and other Caribbean

islands.

Pending a further study looking at the degree of spread of the fungus, chytridiomycosis is

now endemic the Mannophryne olmonae population, rather than epidemic (affecting

other amphibians). It remains possible that increased mortality could result from an

interaction of chytridiomycosis with other factors (Lampo, Rodríguez-Contreras et al.,

2006). The most likely interaction, with temperature, would however act against

increasing severity of the disease. Chytridiomycosis is both more prevalent and more

dangerous at lower temperatures in the laboratory and natural environments (Berger et

al., 2004; Piotrowski et al., 2004; Drew et al., 2006). Thus although global warming

might increase the susceptibility of montane amphibians by bringing their environments

within the optimum temperature range of B. dendrobatidis (Pounds et al., 2006), this is

unlikely in the lowland and lower-montane frog M. olmonae. This species was associated

with a range of water temperatures in both isolated pools (Table 5-8) and in rivers and

streams (Table 5-9), already slightly above the optimum for B. dendrobatidis (17-25ºC;

Piotrowski et al., 2004), and the altitude of the highest peak in Tobago is only 549 m

(Murphy, 1997). By itself, any increase in environmental temperature would seem to

make epidemic chytridiomycosis less rather than more likely in M. olmonae and other

amphibians in Tobago. Increased susceptibility to chytridiomycosis from climate change

is unlikely in amphibian populations in Tobago, as this island does not have high

montane environments, but remains a possibility in the sister island of Trinidad

29

7 Measures for Conservation

7.1 Future of Mannophryne olmonae

The survey highlighted the importance of first order steams and watershed to M.

olmonae. Currently, M. olmonae is afforded protection, under the umbrella designation

of the Main Ridge Forest as a forest reserve. However the relatively low numbers of

frogs recorded is cause for concern. Very little is known about the exact niche (role that

M. olmonae plays in the environment) or its biology. In order to effectively manage this

species, further study is required to monitor the population status and trends of this

species.

The only major threat facing the frog is the frog disease chytridiomycosis, (a skin disease

caused by Batrachochydium dendrobatidis). B. dendrobatidis has been implicated as the

cause of frog decline in Australia, New Zealand, Central America countries and

elsewhere (Berger et al., 1998, Berger et al., 1999) (see Section 1.4). This is the first

know incidence of the fungus in Tobago, also raises conservation concerns for the other

amphibian fauna on the Trinidad and Tobago. The species of most concern in this

respect would be the montane golden tree frog (Phyllodytes auratus) of Trinidad, which

occurs only above 800 m (Murphy, 1997). A survey of chytridiomycosis in Trinidad, and

preventing the possible introduction of B. dendrobatidis to high altitude sites in that

island, is clearly a key requirement for conservation of amphibians in Trinidad and

Tobago.

The exact cause of the population decline of M. olmonae is not known, and maybe as a

result of one cause or a synergy of causes. However, this does not negate the fact that the

species is need of conservation. This need has been recognised by the Environmental

Management Authority, where it has designated M. olmonae 5th on the list of

Environmentally Sensitive Species to be addressed by the EMA for 2007

30

7.2 Education and Public Awareness programme

Project L.E.A.P. has made conservation education is a major aim of this project and

continues to work with schools. To date the Project LEAP has been involved several

formal and informal education programmes and workshops where the theme has been one

of conservation of biodiversity. The target demographic so far has been 18 and under,

(typical demographic found within the group found within primary and secondary

schools). Wider audiences have been targeted through various public symposiums on the

state of vanishing species in Trinidad and Tobago. It is hope that other conservation

based interest groups and stakeholders will get involved in this endeavour.

A series of posters has also been developed and distributed to schools and through the

Environmental Management Authority about the Bloody Bay Poison frog, local

biodiversity in Trinidad and Tobago, threats facing biodiversity and how everyday

citizens can assist in conservation of the natural environment

During the course of this study, presentations have been held a various youth summer

camps and schools on both Trinidad and Tobago. A volunteer programme of the group

has proved to be quite successful. Project LEAP has so far recruited and trained 16

volunteers in the basics of conducting amphibian studies ecological surveys. Some of

these were even teacher and have incorporated conservation education into their teaching

programmes.

7.3 Follow up Activities

The follow-up activities will include the following:

1. Annual monitoring of M. olmonae population as well as further screening on the

frog to determine the extent of spread of B. dendrobatidis. Currently, remaining

funds from the project (see Appendix E) are being used to fund additional B.

dendrobatidis screening on Trinidad.

31

2. Conservation actions will focus on different awareness-raising programs to reach

all groups in the communities where the frogs are found and all the stakeholders

that use the resources (radio, TV, etc).

3. Strengthening and capacity building of park wardens for working with

amphibians, conducting population surveys and B. dendrobatidis screening

4. Recommending the establishment of amphibian conservation zones within the

MRFR, which should ensure the protection of key areas within the MRFR.

Further, these designations should afford protect to the watersheds and the fragile

tributaries that make up these watersheds where the frog is found. Many areas of

frog habitat under direct human impact and are readily accessed. These activities

should be regulated.

32

Acknowledgements: Fieldwork was supported by a 2006 Future Conservationist Award

from the Conservation Leadership Programme (Mannophryne olmonae: An ecological

study in Tobago - Project L.E.A.P.), and seed grants from Conservation International.

We thank Dave Hardy for information on locations of M. olmonae, Giancarlo “Gian”

Lalsingh and Pat Turpin of Environment Tobago for accommodation and logistical help,

numerous assistants for help with fieldwork, and three referees for useful comments.

Handling frogs was under permit from the Wildlife Section of the Forestry Division of

Trinidad and Tobago, and the Department of Natural Resources and the Environment of

the Tobago House of Assembly, courtesy of Nadra Nathai-Gyan and Angela Ramsey,

respectively.

33

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APPENDIX A

TADPOLE KEY

Simplified key to identify Mannophryne tadpoles (2) (Modified from Kenny, 1969) Mannophryne Others a) Tail shape Tapered Rounded (e.g. Bufo marinus) b) Tail colour Prominent spotting Unspotted or diffuse pigment c) Spiracle Sinistral (left side) Ventral or rectal d) Eyes Dorsolateral Lateral e) Dorsal : Ventral fin width 1:1 Not 1:1 f) Upper lip 2 tooth rows 1, 3 or 4 tooth rows g) Lower lip 3 tooth rows 4 tooth rows

APPENDIX B

Batrachochytrium dendrobatidis sampling

Methods for obtaining Batrachochytrium dendrobatidis (BD) samples for PCR testing

Revised 2004 by Lauren J. Livo

Department of Integrative Physiology, CB-354 University of Colorado Boulder, CO 80309-0354 Email: [email protected]

General considerations In terms of PCR samples, bleach and flame destroy DNA, while alcohol preserves DNA. Collection of samples for PCR testing requires that equipment used to collect samples not inhibit DNA detection while also not contaminating the current sample with DNA from a previous sample. In addition, equipment should be decontaminated so that it does not spread BD (or other pathogens) from one animal to another. The PCR sample collection methods described here have been used for obtaining samples from Colorado amphibians for BD (Batrachochytrium dendrobatidis) detection. These methods should apply generally to collection of samples for BD testing from amphibians. Steps for obtaining samples Collect animals Animals should be collected with clean, decontaminated equipment, individually handled with fresh disposable gloves, and placed in individual containers prior to obtaining the samples. Although using Purell or other hand decontamination solutions may prevent the spread of live BD from one animal to another, it is likely to allow contamination of samples with BD DNA (in other words, if you handle a BD-negative animal after handling a BD-positive animal, the PCR samples you obtain may both appear to be positive for BD). Do not place multiple animals in the same container prior to sampling. In this situation, a single infected animal could infect others, and PCR tests could have inflated numbers of positive test results. Equipment (such as individual containers for holding animals) can be cleaned and bleached so that they can be reused. However, this equipment must be rinsed well and allowed to dry prior to reuse so that there is no residual bleach (note that even parts per million bleach in/on/around a sample could possibly destroy all of the DNA in a sample over the course of a few weeks.) Obtaining samples Skin swabs or skin scrapes are the preferred methods of collecting samples from live individuals as the same individual can be tested repeatedly over time. Skin swabs appear to be more gentle to the animal than skin scrapes. Both skin swabs and skin scrapes have similar rates of false negative results when tested on known BD-positive individuals (2 of 15 skin swab and 3 of 15 skin scrape false negative rates in one experiment). Obtain the PCR sample before doing other procedures with the animal (for example, before weighing, checking PIT tags, and so on). Samples require the following equipment:

• Swabs or pointed sticks:

o Swabs: use cotton swabs on 2mm-diameter wood without adhesive (such as Puritan Cotton-Tipped Applicators, #VWR 10806-005, or equivalent) cut to lengths (ca. 3-cm) that fit into 2-ml tubes.

o Skin scrapes: small (ca. 3-cm) lengths of wooden applicators cut so one end is

pointed (6-inch long, 2mm-diameter wooden Puritan Applicators, cut into about 3-cm lengths, VWR catalog # 10805-018, or equivalent)

• 2-ml screw-cap tubes containing 1 ml of 70 percent ethanol (2.0 ml screw cap tube with

cap/500 per bag, VWR catalog # 20170-217). (Note: Although other tubes are available, the VWR tubes have been very good at avoiding leakage.)

To obtain the sample, hold the animal (using fresh gloves) in one hand, and gently but firmly swab (with the cotton swab) or scrape (using the pointed end of the stick) the ventral surface 25 times; for large animals, you may swab or scrape the ventral surface 20 times and the feet and webbing 5 times.

Figure 1. Swabbing ventral surface of amphibian.

Figure 2. Scraping ventral surface of amphibian.

Place the swab (cotton side down) or stick (pointed end down) in the tube. Secure the lid and place in a rack or other container so that the tube remains upright. (Leakage from one tube with BD may get on other tubes and result in contamination of your samples.)

Figure 3. Insert swab or stick into tube with sample at bottom of tube. Other skin tissues (such as toe clip samples or samples of ventral skin from dead animals) may also be collected for PCR testing. Use fine scissors to obtain the tissue. Between each sample, clean the scissors with an ethanol-soaked swab or tissue, and then hold the blades over an open flame to destroy any DNA from the previous sample. Place each sample in a 2 ml tube containing 1 ml of 70 percent ethanol.

Figure 4. Cleaning scissor blades with alcohol.

Figure 5. Passing scissor blades through flame to destroy residual DNA. Toe clipping: If you collect toe clips from live individuals, use fine scissors to amputate the toe tip. When selecting a toe to amputate, you should avoid especially important digits such as the thumb, to avoid having an undue effect on the ability of the animal to feed, reproduce, and so on. Toe clips have rates of false-negative results similar to skin scrapes, but may have more potential for false-positive results through contamination. To obtain a toe clip, cut off the toe tip with the scissors. If bone protrudes from the wound, trim the bone further back (preferably to a joint) so that skin covers the wound, then dab a drop of Vet-bond or other sealant on the wound. In my study, I selected the right rear toe, and continued to encounter individuals with this digit missing 2 to 3 years after the initial sampling. Labeling samples Label each tube with a unique sample number. Other information that should be associated with each sample includes:

• solution (for example, 70 percent ethanol)

• sample type (for example, skin swab, skin scrape, pelvic patch sample, or toe clip)

Figure 6. Label on tube.

Do not place sample information inside the tube. (It can be difficult to extricate, may contaminate other samples through handling, and as paper may contain bleaching agents, may inhibit detection of the target DNA). Designate a plastic bag for the disposal of gloves and other materials (for example, alcohol wipes) to minimize the possibility of contamination. Shipping samples Samples to be shipped should be in a box or mailing tube, not a bubble wrap envelope. The Post Office canceling machine can exert enough force on samples in an envelope to cause the tubes to leak. Acknowledgments Seanna Annis (Department of Biological Sciences, University of Maine, Orono, Maine 04469) helped develop initial sampling techniques, which were further refined by discussions with John Wood (Pisces Molecular, 2200 Central Avenue, Suite F, Boulder, CO 80301). Alex Hyatt (Australian Animal Health Laboratory, Commonwealth Scientific Industrial Research Organization, Geelong, Victoria, Australia) recommended using swabs for PCR sampling of amphibians for Batrachochytrium dendrobatidis. Cynthia Carey (Dept. Integrative Physiology CB-354, University of Colorado, Boulder, CO 80309-0354) provided amphibians used in some laboratory evaluations of methods. Funding for PCR testing was provided by Great Outdoors Colorado through a grant to the Colorado Division of Wildlife.

APPENDIX C

Raw Data

raw data

C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14ID Code River Code Stream order Frog Present Tributary present River Width River Depth velocity pH temperature DO Bank Width Dist. To forest edge % canopy cover

1 DR-100 1 3 0 0 4.2000 0.0350 0.1356 8.59 27.30 7.32 1.90 3.650 80.0 2 DR-150 1 3 0 0 1.1200 0.2700 0.1784 8.69 27.60 7.37 2.30 5.900 20.0 3 DR-200 1 3 0 0 0.5600 0.1400 0.2716 8.72 26.50 7.33 2.55 3.250 80.0 4 DR-250 1 3 1 0 0.5600 0.1500 0.7647 8.75 26.30 5.92 3.70 5.350 85.0 5 DR-300 1 3 1 0 1.8700 0.3600 * 8.74 26.20 7.69 0.00 3.850 60.0 6 DR-350 1 3 1 0 1.6200 0.3400 0.2503 8.68 26.30 7.74 2.25 5.600 50.0 7 DR-400 1 3 1 0 1.7300 0.2900 0.2066 8.30 28.00 6.96 2.55 2.550 45.0 8 DR-450 1 3 1 0 1.2600 0.3500 0.5322 8.45 28.90 6.37 1.15 2.300 90.0 9 DR-600 1 3 0 1 1.1700 0.0400 0.2915 8.48 29.20 6.72 4.00 6.650 0.0 10 DR-650 1 3 1 0 1.8400 0.0500 0.3763 8.52 29.90 6.50 5.10 3.600 0.0 11 DR-700 1 3 1 0 1.7200 0.0400 0.4039 8.26 28.50 6.96 4.05 0.000 10.0 12 DR-750 1 3 0 0 2.0800 0.0500 0.1152 8.44 28.30 6.57 2.60 7.400 40.0 13 DR-800 1 3 0 0 2.7600 0.0400 0.2933 8.46 28.60 6.19 0.00 2.600 80.0 14 DR-850 1 3 1 0 2.4000 0.1400 0.1264 8.47 28.10 6.08 1.70 0.000 95.0 15 DR-900 1 3 0 0 1.2800 0.2800 0.3035 8.51 27.60 6.36 4.30 6.050 90.0 16 DR-950 1 3 1 1 1.5100 0.0900 0.1262 8.53 27.20 6.33 4.70 2.900 35.0 17 DR-1100 1 3 1 1 2.5000 0.0900 0.1143 8.49 26.90 6.24 3.00 2.300 20.0 18 DR-1150 1 3 0 0 1.0500 0.0900 0.4512 8.37 26.70 6.27 0.00 2.750 95.0 19 DR-1200 1 3 0 0 1.3000 0.1200 0.0694 8.23 26.20 5.48 6.45 6.000 40.0 20 DR-1250 1 2 0 0 2.0000 0.0600 0.2783 8.42 26.60 6.27 4.35 3.750 5.0 21 DR-1300 1 2 0 1 3.5000 0.0700 0.2533 8.36 26.90 5.47 0.00 0.000 100.0 22 DR-1350 1 2 1 0 0.6700 0.0600 0.2635 8.45 26.50 6.14 2.20 1.900 60.0 23 DR-1400 1 2 1 0 1.5600 0.2000 0.0721 8.50 27.20 6.11 3.80 4.900 80.0 24 DR-1450 1 2 0 0 0.9000 0.1400 0.1784 8.42 26.90 6.45 0.90 2.950 85.0 25 DR-1500 1 2 1 1 0.8100 0.0700 0.1408 8.37 26.90 6.11 3.50 1.100 65.0 26 DR-1550 1 2 1 1 1.0000 0.1900 0.2279 8.37 27.30 5.88 1.65 1.200 35.0 27 DR-1600 1 2 0 1 1.5800 0.2400 0.0397 8.52 26.30 6.05 0.80 0.900 25.0 28 DR-1650 1 2 0 0 0.9000 0.0600 0.2245 8.55 26.60 6.75 0.00 1.000 80.0 29 DR-1700 1 2 0 0 0.7100 0.1000 0.1589 8.49 26.80 6.06 0.00 0.900 90.0 30 DR-1750 1 2 0 0 0.9000 0.0700 0.0701 8.57 26.80 6.49 1.00 2.700 60.0 31 DR-1800 1 2 1 0 0.9000 0.0800 0.3685 8.54 26.00 6.46 1.85 2.200 90.0 32 DR-1850 1 2 1 0 1.0000 0.0900 0.1153 8.56 27.00 6.00 1.95 3.500 85.0 33 DR-1900 1 2 1 0 0.1200 0.2500 0.1104 8.56 26.20 6.25 0.00 1.750 95.0 34 DR-1950 1 1 1 0 0.9000 0.7000 0.2639 8.50 26.60 6.53 21.45 1.600 90.0 35 DR-2000 1 1 1 1 2.0100 0.1600 0.2261 8.61 26.50 6.84 0.50 2.300 85.0 36 DR-2050 1 1 1 0 0.9000 0.8000 0.0536 8.64 26.40 6.34 0.00 1.100 80.0 37 DR-2100 1 1 0 1 0.6500 0.0000 * 8.49 26.30 6.16 0.00 0.900 85.0

raw data


38 DR-2150 1 1 1 0 0.7000 0.8000 0.0979 8.39 26.10 5.73 0.80 0.800 60.0 39 LD-3100 2 3 0 0 8.6600 0.0900 0.3890 8.66 26.80 7.57 4.65 4.650 90.0 40 LD-3150 2 3 0 0 5.4000 0.1550 0.4260 8.80 27.10 7.43 2.30 2.300 0.0 41 LD-3200 2 3 0 0 5.4000 0.3250 0.2920 8.63 27.15 6.99 0.00 0.000 * 42 LD-3250 2 3 0 0 6.2700 0.2000 0.2030 8.69 27.25 7.29 5.01 5.010 30.0 43 LD-3300 2 3 1 0 5.5000 0.2250 0.2240 8.73 28.00 7.22 5.55 5.550 30.0 44 LD-3350 2 3 0 0 7.6000 0.2700 0.6070 8.64 27.70 7.32 4.65 15.000 15.0 45 LD-3400 2 3 0 0 6.8900 0.2300 0.5550 8.69 27.90 7.56 4.60 4.800 65.0 46 LD-3450 2 3 1 0 10.7000 0.0450 0.4440 8.73 27.90 7.04 2.28 9.000 55.0 47 LD-3500 2 3 0 0 11.6000 0.0800 0.2380 8.62 27.60 7.24 1.10 12.000 0.0 48 LD-3550 2 3 0 0 6.4000 0.0700 0.5180 8.61 27.80 7.26 1.30 5.750 50.0 49 LD-3600 2 3 1 0 6.2000 * 0.3710 8.59 27.65 7.19 1.93 3.050 85.0 50 LD-3650 2 3 0 0 3.5200 0.1500 0.4470 8.43 28.10 7.15 2.10 3.250 80.0 51 LD-3700 2 3 1 0 2.2000 * 0.8610 8.55 29.10 7.22 11.30 11.300 80.0 52 LD-3750 2 3 1 0 3.2000 0.1100 0.7160 8.64 28.25 7.49 5.90 5.900 45.0 53 LD-3800 2 3 1 0 12.3000 0.1600 0.3990 8.57 28.80 6.79 2.20 7.050 0.0 54 LD-3850 2 3 1 0 5.5000 * 0.4430 8.57 28.85 6.47 5.20 8.150 55.0 55 LD-3900 2 3 1 0 3.0000 0.1350 0.2820 8.48 28.65 6.77 2.67 3.700 70.0 56 LD-3950 2 3 0 0 4.6000 0.2500 0.5430 8.61 28.30 7.15 0.70 0.700 80.0 57 LD-4000 2 3 0 0 10.0000 0.0460 0.7110 8.69 28.40 7.62 3.75 3.750 80.0 58 LD-4050 2 3 0 0 9.2000 0.0350 0.5420 8.66 28.75 7.41 4.45 7.750 75.0 59 LD-4500 2 3 0 0 0.0620 0.0700 0.5510 8.64 26.60 7.72 1.70 2.900 20.0 60 LD-4550 2 3 0 0 0.0383 0.2200 0.2770 8.72 26.90 7.08 0.82 2.320 40.0 61 LD-4600 2 3 0 0 0.0450 0.1100 0.6760 8.73 27.00 7.20 0.00 1.500 20.0 62 LD-4650 2 3 0 0 0.0350 0.3300 0.2740 8.72 27.10 7.03 6.10 8.400 85.0 63 LD-4700 2 3 0 0 0.0550 0.3650 0.1330 8.73 27.00 7.23 33.50 5.000 60.0 64 LD-4750 2 3 0 0 0.0373 0.2400 0.5010 8.63 26.95 7.34 0.60 3.335 80.0 65 LD-4800 2 3 1 0 0.0570 0.2500 0.4020 8.68 27.15 7.19 2.10 3.600 30.0 66 LD-4850 2 3 0 0 0.0931 0.1600 0.4360 8.54 27.00 7.02 0.70 6.450 20.0 67 LD-4900 2 3 1 0 0.0870 0.2300 0.2230 8.64 27.00 6.93 0.00 6.150 50.0 68 LD-4950 2 3 0 0 0.1080 0.0900 0.7930 8.62 27.20 7.15 2.40 2.850 40.0 69 LD-5000 2 3 0 0 0.0940 0.0750 0.3070 8.64 27.40 7.01 3.95 3.950 45.0 70 LD-5050 2 3 0 0 0.0830 0.0825 0.2920 8.65 27.25 7.23 0.00 4.000 90.0 71 LD-5100 2 3 1 0 0.0345 0.1700 0.4440 8.68 27.25 7.16 10.25 12.950 95.0 72 LD-5150 2 3 0 0 0.0560 0.4700 0.0630 8.52 27.40 6.86 0.00 0.000 60.0 73 LD-5200 2 3 0 0 0.1350 0.1200 0.4470 8.66 27.35 7.07 1.60 2.700 90.0 74 LD-5250 2 3 0 0 0.0380 0.2600 0.1760 8.63 27.40 6.95 2.10 2.950 90.0

raw data


75 LD-5300 2 3 0 0 0.0600 0.1100 0.4440 8.73 27.70 7.44 2.15 2.150 30.0 76 LD-5350 2 3 0 0 0.0000 0.0750 0.1910 8.75 27.60 7.34 5.50 5.500 60.0 77 LD-5400 2 3 0 0 0.0630 0.1250 0.3240 8.74 27.70 7.28 1.20 1.200 25.0 78 LD-5450 2 3 1 0 0.0355 0.1700 0.6390 8.64 26.40 7.14 1.57 1.570 35.0 79 LD-5500 2 3 0 0 0.0650 0.1000 0.6690 8.55 26.70 7.02 5.32 5.320 55.0 80 LD-5550 2 3 1 0 0.0547 * 0.3450 8.59 26.80 7.11 1.69 1.690 60.0 81 LD-5600 2 3 0 0 0.0295 0.1200 0.1310 8.60 26.90 6.94 9.62 18.850 80.0 82 LD-5650 2 3 0 0 0.1477 0.1400 0.7750 8.26 26.80 7.46 3.00 3.000 5.0 83 LD-6250 2 3 0 0 0.0680 0.0000 0.4040 8.70 27.15 7.18 0.00 0.000 25.0 84 LD-6300 2 3 0 0 0.0700 0.1000 0.7070 8.67 27.55 6.77 0.00 4.500 20.0 85 LD-6350 2 3 0 0 0.0370 0.1500 0.5020 8.62 27.00 7.28 0.90 12.100 25.0 86 LD-6400 2 3 0 0 0.1100 0.0600 0.6760 8.73 27.10 7.13 0.00 19.550 40.0 87 LD-6450 2 3 0 0 0.0600 0.2400 0.2490 8.55 26.90 7.22 1.90 1.900 30.0 88 LD-6500 2 3 0 0 0.0725 0.1500 0.4630 * 26.90 7.10 * 0.000 1.0 89 LD-6550 2 3 1 0 0.0690 0.1600 0.4230 * 26.90 7.11 * 4.300 50.0 90 LD-6600 2 3 0 0 0.0540 0.1200 0.3340 * 27.00 7.03 * 7.000 50.0 91 LD-6650 2 3 0 0 0.0770 0.1200 0.5210 * 27.00 6.99 * 2.900 40.0 92 LD-6700 2 3 0 0 0.0475 0.1300 0.5160 * 27.30 6.88 * 8.600 1.0 93 LD-6750 2 3 1 0 0.0865 0.1300 0.1950 * 27.10 7.10 * 6.200 20.0 94 LD-6800 2 3 1 0 0.0400 0.2100 0.5380 * 27.10 6.86 * 0.000 0.0 95 LD-6850 2 3 0 0 0.0330 0.2200 0.4690 * 27.20 6.84 * 0.000 70.0 96 LD-6900 2 3 0 0 0.0350 0.1700 0.3760 * 27.20 6.79 * 0.000 0.0 97 LD-6950 2 3 1 0 0.0320 0.1600 0.4310 * 27.20 7.01 * 5.100 20.0 98 LD-7000 2 3 1 0 0.0340 0.2100 0.4380 * 27.30 6.75 * 4.100 1.0 99 LD-7050 2 3 0 0 0.0515 0.1400 0.7550 * 27.50 7.14 * 0.000 10.0 100 LD-7100 2 3 0 0 0.0400 0.2500 0.1510 * 27.60 7.10 * 3.600 30.0 101 LD-7150 2 3 0 0 0.0570 1.0000 0.0930 * 27.60 7.17 * 1.000 85.0 102 LD-7200 2 3 0 0 0.0240 0.2400 0.6670 * 27.60 7.07 * 7.300 55.0 103 LD-7250 2 3 0 0 0.0690 0.2200 0.4850 * 27.70 7.23 * 2.800 40.0 104 LD-7300 2 3 1 0 0.0755 0.4200 0.3020 * 27.80 7.11 * 3.500 10.0 105 LD-7350 2 3 1 0 0.0395 0.2100 0.5930 * 27.80 7.50 * 0.000 0.0 106 LD-7400 2 3 1 0 0.0310 0.4000 0.6250 * 28.00 7.57 * 7.550 0.0 107 LD-7450 2 3 1 0 0.0985 0.3000 0.2300 * 27.90 7.83 * 0.000 0.0 108 LD-7500 2 3 1 0 0.0520 0.3000 0.3340 * 27.80 7.55 * 9.200 0.0 109 LD-7550 2 3 1 0 0.0620 0.2000 0.4940 * 27.70 7.47 * 4.400 0.0 110 LD-7600 2 3 0 0 0.0760 0.3300 0.2300 * 27.30 7.54 * 4.500 0.0 111 LD-7650 2 3 1 0 0.0520 0.3700 0.2040 * 27.30 7.49 * 8.500 0.0

raw data


112 LD-7700 2 3 0 0 0.0500 0.1900 0.3160 * 27.50 7.40 * 5.750 0.0 113 LD-7750 2 3 0 0 0.0600 0.3000 0.2360 * 22.30 7.57 * 2.000 0.0 114 LD-7800 2 3 0 0 0.0430 0.1900 0.7020 * 27.30 7.39 * 5.750 10.0 115 LD-7850 2 3 0 0 0.0580 0.2000 0.4380 * 26.10 7.57 * 7.100 1.0 116 BB-1350 3 3 0 0 8.9000 0.0700 0.2670 8.24 26.80 7.67 0.75 4.210 60.0 117 BB-1400 3 3 0 0 6.5000 0.1900 0.3280 8.18 26.15 6.80 2.80 4.800 25.0 118 BB-1450 3 3 0 0 3.4000 * 0.1970 8.16 26.40 7.10 1.55 4.325 20.0 119 BB-1500 3 3 0 0 6.6000 0.1200 0.3800 8.26 27.15 6.85 16.40 11.625 0.5 120 BB-1550 3 3 0 0 4.5700 0.1300 0.4050 8.21 26.70 6.40 21.05 13.725 10.0 121 BB-1600 3 3 1 0 11.7000 0.2200 0.0520 8.27 26.90 6.36 5.90 6.130 40.0 122 BB-1650 3 3 0 0 6.2000 0.1400 0.2970 8.36 26.70 6.89 2.80 4.845 5.0 123 BB-1700 3 3 0 0 7.2000 0.2100 0.3470 8.27 27.00 7.23 5.85 6.540 0.0 124 BB-1750 3 3 0 0 4.8000 0.1400 0.5100 6.69 27.10 8.24 1.75 4.995 5.0 125 BB-1800 3 3 0 0 7.4000 0.6700 0.1610 8.26 27.05 6.77 0.00 3.385 10.0 126 BB-1850 3 3 0 0 3.6000 0.7800 0.1730 8.28 27.15 6.70 25.00 * 70.0 127 BB-3350 3 3 0 1 3.5500 1.2000 0.1300 8.65 25.35 7.65 0.75 4.200 70.0 128 BB-3400 3 3 0 0 1.5900 0.2700 0.5620 * 25.60 7.65 9.60 8.625 40.0 129 BB-3450 3 3 0 0 10.8000 0.6100 0.1750 * 2.51 7.57 0.00 3.785 55.0 130 BB-3500 3 3 0 0 9.0000 0.1400 0.1880 * 26.30 7.23 3.25 5.240 50.0 131 BB-3550 3 3 0 0 5.2000 1.2000 0.1750 * 25.70 7.57 3.10 5.335 85.0 132 BB-3600 3 3 0 0 6.0000 0.5300 0.0830 * 26.00 7.68 0.00 3.840 30.0 133 BB-3650 3 3 1 0 2.8300 0.2300 0.3110 * 25.80 7.63 1.00 4.315 60.0 134 BB-3700 3 3 0 0 5.8700 0.9300 0.0950 * 24.90 8.23 3.87 6.048 60.0 135 BB-3800 3 3 0 0 9.7000 0.3000 0.7320 * 25.30 7.62 0.50 4.060 80.0 136 BB-3850 3 3 1 0 8.4000 0.2100 0.3610 * 25.00 7.65 0.00 3.825 40.0 137 BB-3900 3 3 0 0 9.8000 0.3400 0.3230 * 25.20 7.57 0.00 3.785 60.0 138 BB-3950 3 3 0 0 8.5000 0.2200 0.2400 * 25.30 7.56 0.00 * 50.0 139 BB-4000 3 3 1 0 9.2000 0.2500 0.1500 * 25.20 7.65 0.00 * 25.0 140 BB-4050 3 3 1 0 12.7000 0.2700 0.6880 * 25.20 7.50 0.00 * 70.0 141 BB-4100 3 3 0 0 7.6000 0.3500 0.1690 * 25.20 7.45 0.00 * 30.0 142 RR-2650 4 2 0 0 4.8500 0.2000 0.4900 * 26.10 7.37 * 5.100 35.0 143 RR-2700 4 2 0 0 8.2000 0.1600 0.5000 * 26.70 6.84 * 0.900 10.0 144 RR-2750 4 2 0 0 8.1000 0.1700 0.6500 * 26.60 6.90 * 0.450 5.0 145 RR-2800 4 2 0 0 7.7500 0.2000 0.3050 * 26.60 6.95 * 0.300 15.0 146 RR-2850 4 2 0 0 0.6700 0.2950 0.3800 * 26.30 7.25 * 6.420 10.0 147 RR-2900 4 2 0 1 5.5300 0.2050 0.3750 * 26.50 7.23 * 0.000 45.0 148 RR-2950 4 2 0 1 4.1000 0.1600 0.5950 * 26.70 7.21 * 1.750 85.0

raw data


149 RR-3000 4 2 0 0 5.5000 0.1750 0.3050 * 26.30 7.30 * 0.420 90.0 150 RR-3050 4 2 0 0 8.5000 0.9000 0.2250 * 25.70 7.98 * 4.040 95.0 151 RR-3100 4 2 0 0 3.7800 0.2500 0.4000 * 25.90 8.11 * 3.000 80.0 152 RR-3150 4 2 0 0 2.7700 0.3800 0.2300 * 25.70 7.98 * 0.000 95.0 153 RR-3200 4 2 0 0 4.3000 0.2750 0.1450 * 25.80 7.82 * 1.500 35.0 154 RR-3250 4 2 1 1 5.6000 0.5000 0.1200 * 25.60 7.78 * 5.600 35.0 155 RR-3300 4 2 0 0 3.1900 0.2800 0.4350 * 25.50 8.12 * 0.000 45.0 156 RR-3350 4 2 0 0 9.2600 0.2500 0.2050 * 25.70 7.82 * 2.650 40.0 157 RR-3400 4 2 0 1 4.4000 0.3200 0.4450 * 25.60 7.65 * 9.400 25.0 158 RR-3450 4 2 0 0 7.3000 0.6750 0.7250 * 25.50 7.78 * 1.000 15.0 159 RR-3500 4 2 1 0 3.4400 0.2000 0.3150 * 25.70 7.73 * 2.500 10.0 160 AG-0 6 2 1 0 0.0660 0.5800 0.2930 8.74 25.25 8.17 7.25 6.950 50.0 161 AG-50 6 2 1 0 0.0850 0.2000 0.1750 8.57 25.30 7.91 4.77 7.080 60.0 162 AG-100 6 2 0 0 0.0700 0.3940 * 8.55 25.50 7.95 7.13 7.135 60.0 163 AG-150 6 2 0 0 0.0152 1.5000 0.5630 7.41 25.20 7.94 8.01 28.500 70.0 164 AG-200 6 2 0 1 0.0900 0.6300 0.1930 8.56 25.50 7.71 4.75 6.470 20.0 165 AG-250 6 2 0 0 0.0230 0.3800 0.3530 8.54 25.40 7.64 0.00 4.215 20.0 166 T2-50 7 1 1 0 0.8000 0.0650 0.1930 5.74 26.00 7.25 3.20 3.200 15.0 167 T2-100 7 1 0 0 0.8800 0.0600 0.1820 5.69 26.00 6.95 2.52 2.520 70.0 168 T2-150 7 1 0 0 0.7550 0.1100 0.1420 5.45 26.05 6.92 3.50 3.500 65.0 169 T2-200 7 1 0 0 0.7800 0.1200 0.1690 5.53 25.95 7.64 1.40 1.400 30.0 170 T2-250 7 1 1 0 0.6100 0.1700 0.2330 5.28 25.90 7.64 1.30 1.300 60.0 171 T2-300 7 1 1 0 0.1500 0.0530 0.0310 5.32 25.75 7.59 1.00 1.000 80.0 172 T2-350 7 1 0 0 0.5100 0.0700 0.2780 5.06 25.75 7.10 1.00 1.000 60.0 173 T2-400 7 1 0 0 1.0000 0.0400 0.3210 4.99 26.35 7.38 2.50 2.500 85.0 174 T4-50 8 1 1 0 0.1700 0.0300 * 7.25 26.40 7.60 4.00 4.000 40.0 175 T5-50 9 1 0 0 1.4200 0.2700 * 6.79 26.90 7.34 6.80 0.000 30.0 176 T5-100 9 1 1 0 0.7500 0.1500 0.0530 6.61 27.05 5.24 4.20 0.000 50.0 177 T5-150 9 1 0 0 1.1000 * 0.2370 6.50 26.30 7.00 1.20 0.000 60.0 178 T5-200 9 1 0 0 3.0000 0.0800 0.4360 6.47 25.75 7.50 0.00 0.000 50.0 179 T5-250 9 1 0 0 0.1200 0.3700 0.2160 6.37 25.60 7.71 0.00 0.000 80.0 180 T18-0 10 1 1 0 0.3000 0.0000 * * * * 0.80 0.800 50.0 181 T18-50 10 1 1 0 0.3000 0.0000 * * * * 0.50 0.500 80.0 182 T20-0 11 1 1 0 1.5000 0.0000 * * * * 0.00 3.600 40.0 183 T20-50 11 1 1 0 0.3000 0.0000 * * * * 0.50 1.500 60.0 184 T20-100 11 1 1 1 0.3000 0.0000 * * * * 0.00 0.000 80.0 185

————— 20/06/2007 09:37:58 PM ————————————————————

Binary Logistic Regression: Frog Present versus Stream order; Tributary pr; ...

Link Function: Logit

Response Information

Variable Value CountFrog Pre 1 44 (Event) 0 118 Total 162

162 cases were used 22 cases contained missing values

Logistic Regression Table Odds 95% CI

Predictor Coef SE Coef Z P Ratio Lower UpperConstant -12.806 8.751 -1.46 0.143Stream o -0.3832 0.3908 -0.98 0.327 0.68 0.32 1.47Tributar 1 0.4062 0.6604 0.62 0.539 1.50 0.41 5.48

River Wi -0.04555 0.06449 -0.71 0.480 0.96 0.84 1.08River De 1.3324 0.9155 1.46 0.146 3.79 0.63 22.80velocity -1.809 1.222 -1.48 0.139 0.16 0.01 1.80temperat 0.7042 0.2976 2.37 0.018 2.02 1.13 3.62DO -0.8265 0.4078 -2.03 0.043 0.44 0.20 0.97Dist. To 0.03784 0.05032 0.75 0.452 1.04 0.94 1.15% canopy -0.004133 0.006556 -0.63 0.528 1.00 0.98 1.01

Log-Likelihood = -82.391Test that all slopes are zero: G = 24.710; DF = 9; P-Value = 0.003

Goodness-of-Fit Tests

Method Chi-Square DF PPearson 183.545 152 0.041Deviance 164.781 152 0.226Hosmer-Lemeshow 9.214 8 0.325

Table of Observed and Expected Frequencies:(See Hosmer-Lemeshow Test for the Pearson Chi-Square Statistic)

GroupValue 1 2 3 4 5 6 7 8 9 10 Total1 Obs 2 2 2 2 2 3 3 10 7 11 44 Exp 0.9 1.7 2.3 2.9 3.7 3.9 4.7 5.7 7.4 10.9

0 Obs 14 14 14 14 15 13 13 6 9 6 118 Exp 15.1 14.3 13.7 13.1 13.3 12.1 11.3 10.3 8.6 6.1

Total 16 16 16 16 17 16 16 16 16 17 162

Measures of Association:(Between the Response Variable and Predicted Probabilities)

Pairs Number Percent Summary MeasuresConcordant 3869 74.5% Somers' D 0.50Discordant 1293 24.9% Goodman-Kruskal Gamma 0.50Ties 30 0.6% Kendall's Tau-a 0.20Total 5192 100.0%

Saving file as: C:\Program Files\MTBWIN\Data\frog3.MPJ* NOTE * Existing file replaced.








Predictor Coef SE Coef Z P Ratio Lower UpperConstant -0.3823 0.9372 -0.41 0.683Stream o 0.1067 0.3019 0.35 0.724 1.11 0.62 2.01Tributar 1 0.6154 0.6008 1.02 0.306 1.85 0.57 6.01

River Wi -0.07500 0.06180 -1.21 0.225 0.93 0.82 1.05River De -0.1493 0.8037 -0.19 0.853 0.86 0.18 4.16velocity -2.387 1.147 -2.08 0.038 0.09 0.01 0.87Dist. To 0.01795 0.05057 0.35 0.723 1.02 0.92 1.12% canopy -0.000698 0.006215 -0.11 0.911 1.00 0.99 1.01






0 Obs 12 15 14 14 11 11 12 11 7 11 118 Exp 14.1 13.3 12.8 12.4 12.7 11.6 11.1 10.7 10.0 9.2

Total 16 16 16 16 17 16 16 16 16 17 162










River Wi -0.07841 0.06053 -1.30 0.195 0.92 0.82 1.04River De -0.1350 0.7710 -0.18 0.861 0.87 0.19 3.96Dist. To 0.00163 0.04763 0.03 0.973 1.00 0.91 1.10% canopy 0.001674 0.005829 0.29 0.774 1.00 0.99 1.01






0 Obs 13 16 10 9 13 16 16 10 9 10 122 Exp 14.2 13.6 12.7 12.3 12.9 12.1 11.9 11.4 10.8 10.2

Total 17 17 17 17 18 17 17 17 17 18 172



Saving file as: C:\Program Files\MTBWIN\Data\frog3.MPJ* NOTE * Existing file replaced.Saving file as: C:\Program Files\MTBWIN\Data\frog3.MPJ* NOTE * Existing file replaced.

Binary Logistic Regression: Frog Present versus Stream order; Tributary pr; ...* NOTE * Algorithm has not converged after 20 iterations. * Convergence has not been reached for the

* parameter estimates criterion. * The results may not be reliable. * Try increasing the maximum number of iterations.







River Wi 0.00948 0.08486 0.11 0.911 1.01 0.85 1.19River De 0.655 1.078 0.61 0.543 1.93 0.23 15.92Dist. To 0.01735 0.05600 0.31 0.757 1.02 0.91 1.14% canopy -0.007509 0.007182 -1.05 0.296 0.99 0.98 1.01River Co 2 -0.6889 0.6899 -1.00 0.318 0.50 0.13 1.94 3 -0.896 1.166 -0.77 0.442 0.41 0.04 4.01 4 -2.699 1.377 -1.96 0.050 0.07 0.00 1.00 6 -0.155 1.443 -0.11 0.914 0.86 0.05 14.48 7 -22 9944 -0.00 0.998 0.00 0.00 * 9 -1.841 1.660 -1.11 0.267 0.16 0.01 4.10

velocity -1.666 1.318 -1.26 0.206 0.19 0.01 2.50temperat 0.6130 0.3496 1.75 0.080 1.85 0.93 3.66DO -0.1576 0.5090 -0.31 0.757 0.85 0.31 2.32






0 Obs 16 13 15 15 15 14 8 8 7 7 118 Exp 15.8 15.0 14.1 13.4 13.5 12.2 11.3 10.0 7.8 5.0

Total 16 16 16 16 17 16 16 16 16 17 162


Pairs Number Percent Summary MeasuresConcordant 4132 79.6% Somers' D 0.59

Discordant 1047 20.2% Goodman-Kruskal Gamma 0.60Ties 13 0.3% Kendall's Tau-a 0.24Total 5192 100.0%


————— 21/06/2007 08:24:59 AM ————————————————————

Welcome to Minitab, press F1 for help.Retrieving project from file: C:\Program Files\MTBWIN\Data\frog3.MPJ

————— 26/06/2007 07:37:08 PM ————————————————————


Results for: frog.MTW

Regression Analysis: Frog Present versus temperature

The regression equation isFrog Present = - 0.374 + 0.0266 temperature

179 cases used 5 cases contain missing values

Predictor Coef SE Coef T PConstant -0.3741 0.4563 -0.82 0.413temperat 0.02657 0.01704 1.56 0.121

S = 0.4715 R-Sq = 1.4% R-Sq(adj) = 0.8%

Analysis of Variance

Source DF SS MS F PRegression 1 0.5403 0.5403 2.43 0.121Residual Error 177 39.3479 0.2223Total 178 39.8883

Unusual ObservationsObs temperat Frog Pre Fit SE Fit Residual St Resid129 2.5 0.0000 -0.3074 0.4137 0.3074 1.36 X

X denotes an observation whose X value gives it large influence.

Regression Analysis: Frog Present versus DO

The regression equation isFrog Present = 1.46 - 0.158 DO


Predictor Coef SE Coef T PConstant 1.4559 0.4419 3.29 0.001DO -0.15752 0.06192 -2.54 0.012

S = 0.4663 R-Sq = 3.5% R-Sq(adj) = 3.0%



Unusual ObservationsObs DO Frog Pre Fit SE Fit Residual St Resid19 5.48 0.0000 0.5927 0.1071 -0.5927 -1.31 X21 5.47 0.0000 0.5943 0.1076 -0.5943 -1.31 X38 5.73 1.0000 0.5533 0.0926 0.4467 0.98 X

176 5.24 1.0000 0.6305 0.1212 0.3695 0.82 X


Regression Analysis: Frog Present versus velocity

The regression equation isFrog Present = 0.409 - 0.222 velocity


Predictor Coef SE Coef T PConstant 0.40895 0.07385 5.54 0.000velocity -0.2215 0.1892 -1.17 0.243

S = 0.4723 R-Sq = 0.8% R-Sq(adj) = 0.2%



Unusual ObservationsObs velocity Frog Pre Fit SE Fit Residual St Resid 4 0.765 1.0000 0.2395 0.0878 0.7605 1.64 X51 0.861 1.0000 0.2182 0.1047 0.7818 1.70 X68 0.793 0.0000 0.2333 0.0927 -0.2333 -0.50 X82 0.775 0.0000 0.2373 0.0895 -0.2373 -0.51 X


Saving file as: C:\Program Files\MTBWIN\Data\frog4.MPJ

————— 10/09/2007 07:29:58 PM ————————————————————


APPENDIX D

PCR Results

Pisces Molecular LLC 9/17/2007

Chytrid Fungus Test Results PCR assay for B. dendrobatidis

BP Conservation Project - Tobago November, 2006 samples

Test samples:

One hundred twenty five skin swab samples were received 11/20/06 from Jahson Berhane. Note one sample vial (T20-01, Pisces # 67775) did not contain a swab but only a small amount of liquid.

Sample Preparation: For each sample 800uL to 1 ml of 70% ethanol was added to the sample vial, mixed by pipetting the

liquid up and down, then the entire volume, including any visible skin pieces, was transferred to a microfuge tube. After spinning at maximum speed in a microcentrifuge (~16,000 x G) for 3 minutes, the supernatant was drawn off and discarded, tissue lysis buffer added, and any pellet resuspended by vortexing. 10 ug of carrier DNA was added to the lysis buffer for the skin swab samples (no carrier was added to the egg mass samples). Total DNA was extracted from all samples using a spin-column DNA purification procedure.

PCR assay: All sample DNA preparations were assayed for the presence of the Batrachochytrium dendrobatidis ribosomal RNA Intervening Transcribed Sequence (ITS) region by 45 cycle single-round PCR amplification using an assay developed by Seanna Annis and modified for greater specificity and sensitivity at Pisces. Each PCR run included the following controls: Positive DNA: DNA prepared from a laboratory culture of B. dendrobatidis, Strain JEL 270, kindly

provided by Joyce Longcore. This sample was previously demonstrated to be positive by PCR. The signal from this sample is the standard for a strong positive (++) signal.

Negative DNA: DNA prepared from a laboratory culture of a non-batracho chytrid fungus, Strain JEL 151, kindly provided by Joyce Longcore This sample was previously demonstrated to be negative (-) by PCR.

No DNA: H2O in place of template DNA. This reaction remains uncapped during addition of sample DNA to the test reactions, and serves as a control to detect contaminating DNA in the PCR reagents or carryover of positive DNA during reaction set-up.

Results: Twenty one samples were positive for the B. dendrobatidis ribosomal RNA ITS region; the remaining 104 swab samples were negative. Individual sample results are shown on the following four pages.

Scoring: +++ = very strong positive signal

++ = strong positive signal + = positive signal w+ = weak positive signal - = no signal/below limit of detection

Individual sample PCR test resultsSource Source ID # Source Information Pisces # Sample Form/Condition Tested For Results Comments

BPC/JB Doctors R #1 L.V. ? 06/8/06 67770 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prep; Hard to read labelBPC/JB Doctors R #2 M.O. 6/8/06 67771 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prep

BPC/JB Doctors R. - 2 M.O. 9-8-06 67663 swab/EtOH B. dendrobatidis - 1ml 70%EtOH added before prepBPC/JB Doctors R. - 3 M.O. 9-8-06 67664 swab/EtOH B. dendrobatidis ++ 1ml 70%EtOH added before prepBPC/JB Doctors R. - 4 M.O. 9-8-06 67665 swab/EtOH B. dendrobatidis - 1ml 70%EtOH added before prepBPC/JB Doctors R. - 5 M.O. 9-8-06 67666 swab/EtOH B. dendrobatidis w+ 1ml 70%EtOH added before prepBPC/JB Doctors R. - 6 M.O. 9-8-06 67667 swab/EtOH B. dendrobatidis - 1ml 70%EtOH added before prepBPC/JB Doctors R. - 7 M.O. 9-8-06 67668 swab/EtOH B. dendrobatidis ++ 1ml 70%EtOH added before prepBPC/JB Doctors R. - 8 M.O. 9-8-06 67669 swab/EtOH B. dendrobatidis +++ 1ml 70%EtOH added before prepBPC/JB Doctors R. - 9 M.O. 9-8-06 67670 swab/EtOH B. dendrobatidis - 1ml 70%EtOH added before prepBPC/JB Doctors R. - 10 M.O. 9-8-06 67671 swab/EtOH B. dendrobatidis - 850uL 70%EtOH added before prepBPC/JB Doctors R. - 11 M.O. 9-8-06 67672 swab/EtOH B. dendrobatidis - 850uL 70%EtOH added before prep

BPC/JB Doctors R. - 12 M.O. 10-8-07 67673 swab/EtOH B. dendrobatidis - 850uL 70%EtOH added before prepBPC/JB Doctors R. - 13 M.O. 10-8-07 67674 swab/EtOH B. dendrobatidis - 850uL 70%EtOH added before prepBPC/JB Doctors R. - 14 M.O. 10-8-07 67675 swab/EtOH B. dendrobatidis - 850uL 70%EtOH added before prepBPC/JB Doctors R. - 15 M.O. 10-8-07 67676 swab/EtOH B. dendrobatidis - 850uL 70%EtOH added before prepBPC/JB Doctors R. - 16 M.O. 10-8-07 67677 swab/EtOH B. dendrobatidis - 850uL 70%EtOH added before prepBPC/JB Doctors R. - 17 M.O. 10-8-07 67678 swab/EtOH B. dendrobatidis + 850uL 70%EtOH added before prepBPC/JB Doctors R. - 18 M.O. 10-8-07 67679 swab/EtOH B. dendrobatidis - 850uL 70%EtOH added before prepBPC/JB Doctors R. - 19 M.O. 10-8-07 67680 swab/EtOH B. dendrobatidis +++ 850uL 70%EtOH added before prepBPC/JB Doctors R. - 20 M.O. 10-8-07 67681 swab/EtOH B. dendrobatidis - 850uL 70%EtOH added before prepBPC/JB Doctors R. - 21 M.O. 10-8-07 67682 swab/EtOH B. dendrobatidis +++ 850uL 70%EtOH added before prepBPC/JB Doctors R. - 22 M.O. 10-8-07 67683 swab/EtOH B. dendrobatidis - 850uL 70%EtOH added before prepBPC/JB Doctors R. - 23 M.O. 10-8-07 67684 swab/EtOH B. dendrobatidis - 850uL 70%EtOH added before prep

BPC/JB Doctors R. - 24 11.8.06 67685 swab/EtOH B. dendrobatidis + 850uL 70%EtOH added before prepBPC/JB Doctors R. - 25 11.8.06 67686 swab/EtOH B. dendrobatidis - 850uL 70%EtOH added before prepBPC/JB Doctors R. - 26 11.8.06 67687 swab/EtOH B. dendrobatidis - 850uL 70%EtOH added before prepBPC/JB Doctors R. - 27 11.8.06 67688 swab/EtOH B. dendrobatidis - 850uL 70%EtOH added before prepBPC/JB Doctors R. - 28 11.8.06 67689 swab/EtOH B. dendrobatidis - 850uL 70%EtOH added before prepBPC/JB Doctors R. - 29 11.8.06 67690 swab/EtOH B. dendrobatidis - 850uL 70%EtOH added before prepBPC/JB Doctors R. - 30 11.8.06 67691 swab/EtOH B. dendrobatidis - 850uL 70%EtOH added before prepBPC/JB Doctors R. - 31A 11.8.06 67692 swab/EtOH B. dendrobatidis - 850uL 70%EtOH added before prepBPC/JB Doctors R. - 31B 11.8.06 67693 swab/EtOH B. dendrobatidis ++ 850uL 70%EtOH added before prepBPC/JB Doctors R. - 32 11.8.06 67694 swab/EtOH B. dendrobatidis - 850uL 70%EtOH added before prepBPC/JB Doctors R. - 33 11.8.06 67695 swab/EtOH B. dendrobatidis +++ 800uL 70%EtOH added before prepBPC/JB Doctors R. - 34 11.8.06 67696 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prepBPC/JB Doctors R. - 35 11.8.06 67697 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prepBPC/JB Doctors R. - 36 11.8.06 67698 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prepBPC/JB Doctors R. - 37 11.8.06 67699 swab/EtOH B. dendrobatidis ++ 800uL 70%EtOH added before prepBPC/JB Doctors R. - 38 11.8.06 67700 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prep

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BPC/JB Doctors R. - 39 11.8.06 67701 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prepBPC/JB Doctors R. - 40 11.8.06 67702 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prepBPC/JB Doctors R. - 41 12.8.06 67703 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prepBPC/JB Doctors R. - 42 12.8.06 67704 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prepBPC/JB Doctors R. - 43 12.8.06 67705 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prepBPC/JB Doctors R. - 44 12.8.06 67706 swab/EtOH B. dendrobatidis ++ 800uL 70%EtOH added before prepBPC/JB Doctors R. - 45 12.8.06 67707 swab/EtOH B. dendrobatidis ++ 800uL 70%EtOH added before prepBPC/JB Doctors R. - 46 12.8.06 67708 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prepBPC/JB Doctors R. - 47 12.8.06 67709 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prepBPC/JB Doctors R. - 48 12.8.06 67710 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prepBPC/JB Doctors R. - 49 12.8.06 67711 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prepBPC/JB Doctors R. - 50 12.8.06 67712 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prepBPC/JB Doctors R. - 51 12.8.06 67713 swab/EtOH B. dendrobatidis ++ 800uL 70%EtOH added before prepBPC/JB Doctors R. - 52 12.8.06 67714 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prepBPC/JB Doctors R. - 53 12.8.06 67715 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prepBPC/JB Doctors R. - 54 12.8.06 67716 swab/EtOH B. dendrobatidis + 800uL 70%EtOH added before prepBPC/JB Doctors R. - 55 12.8.06 67717 swab/EtOH B. dendrobatidis + 800uL 70%EtOH added before prepBPC/JB Doctors R. - 56 12.8.06 67718 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prepBPC/JB Doctors R. - 57 12.8.06 67719 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prepBPC/JB Doctors R. - 58 12.8.06 67720 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prepBPC/JB Doctors R. - 59 12.8.06 67721 swab/EtOH B. dendrobatidis ++ 800uL 70%EtOH added before prepBPC/JB Doctors R. - 60 12.8.06 67722 swab/EtOH B. dendrobatidis +++ 800uL 70%EtOH added before prepBPC/JB Doctors R. - 61 12.8.06 67723 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prepBPC/JB Doctors R. - 62 12.8.06 67724 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prep

BPC/JB Doctors R M. olmonae 08-07-06 67777 swab/EtOH B. dendrobatidis ++ 800uL 70%EtOH added before prepBPC/JB Doctors R M. olmonae 07-07-06 67778 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prepBPC/JB Doctors R M. olmonae 10-07-06 67779 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prepBPC/JB Doctors R M. olmonae 08-07-06 67782 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prepBPC/JB Doctors R M. olmonae 08-07-06 67783 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prepBPC/JB Doctors R 10.7.06 67785 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prep

BPC/JB B. merinus Man O War 30-8-06 67725 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prepBPC/JB B. merinus Man O War 30-8-06 67726 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prepBPC/JB B. merinus Man O War 30-8-06 67727 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prepBPC/JB B. merinus Man O War 30-8-06 67728 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prepBPC/JB B. merinus Man O War 30-8-06 67729 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prepBPC/JB B. merinus Man O War 30-8-06 67730 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prepBPC/JB B. merinus Man O War 30-8-06 67731 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prepBPC/JB B. merinus Man O War 30-8-06 67732 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prepBPC/JB B. merinus Man O War 30-8-06 67733 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prepBPC/JB B. merinus Man O War 30-8-06 67734 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prepBPC/JB B. merinus Man O War 30-8-06 67735 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prep



BPC/JB B. merinus Man O War 30-8-06 67736 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prepBPC/JB B. merinus Man O War 30-8-06 67737 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prepBPC/JB B. merinus Man O War 30-8-06 67738 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prepBPC/JB B. merinus Man O War 30-8-06 67739 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prepBPC/JB B. merinus Man O War 30-8-06 67740 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prepBPC/JB B. merinus Man O War 30-8-06 67741 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prepBPC/JB B. merinus Man O War 30-8-06 67742 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prepBPC/JB B. merinus Man O War 30-8-06 67743 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prepBPC/JB B. merinus Man O War 30-8-06 67744 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prepBPC/JB B. merinus Man O War 30-8-06 67745 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prepBPC/JB B. merinus Man O War 30-8-06 67746 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prepBPC/JB B. merinus Man O War 30-8-06 67747 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prepBPC/JB B. merinus Man O War 30-8-06 67748 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prepBPC/JB B. merinus Man O War 30-8-06 67749 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prepBPC/JB B. merinus Man O War 30-8-06 67750 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prepBPC/JB B. merinus S.C. Road 30-08-06 67762 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prepBPC/JB B. merinus S.C. Road 30-08-06 67763 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prepBPC/JB B. merinus S.C. Road 30-08-06 67764 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prepBPC/JB B. merinus S.C. Road 30-08-06 67765 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prepBPC/JB B. merinus S.C. Road 30-08-06 67766 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prepBPC/JB B. merinus S.C. Road 30-08-06 67767 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prep

BPC/JB M. olmonae Bloody Bay 070806 67753 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prep; Sprayed when cap opened

BPC/JB M. olmonae Argyle 26-08-06 67756 swab/EtOH B. dendrobatidis ++ 800uL 70%EtOH added before prepBPC/JB M. olmonae Argyle 26-08-06 67757 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prepBPC/JB M. olmonae Argyle 26-08-06 67758 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prepBPC/JB M. olmonae Argyle 26-08-06 67759 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prepBPC/JB M. olmonae Argyle 26-08-06 67760 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prep

BPC/JB M.O. #1 Louis Dior 8/8/06 67768 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prepBPC/JB M.O. #2 Louis Dior 8/8/06 67769 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prep

BPC/JB NOR 4.1 T18-01? 67754 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prep; Hard to read label; Sprayed when cap opened

BPC/JB T18 River 18-08-06 67761 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prep; Hard to read labelBPC/JB T18 River - 01 18-08-06 67772 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prepBPC/JB T18 River - 03 18-08-06 67773 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prepBPC/JB T18 River - 04 18-08-06 67774 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prepBPC/JB T20-01 19-8-06 67775 no swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prep; No swab in sample (?), but

a little liquidBPC/JB T20-02 19-8-06 67776 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prep



BPC/JB T20-03 19-08-06 67755 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prep

BPC/JB DR02 7.7.06 67781 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prepBPC/JB DR06 8.11.06 67787 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prep; Hard to read label

BPC/JB Bloody Bay M. olmonae 07-08-06 67784 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prep

BPC/JB Louis Dior 10.7.06 67786 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prep

BPC/JB 1 + 800 01 f/s 67751 swab/EtOH B. dendrobatidis ++ 800uL 70%EtOH added before prep

BPC/JB King's Bay toe clip bag 67752 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prep

BPC/JB RP 3:45? 09.02.06 67780 swab/EtOH B. dendrobatidis - 800uL 70%EtOH added before prep; Hard to read label


APPENDIX E

Financial Report

TTD USD TTD USD TTD USDPhase 1: Project PreparationAdministrative TT$8,316.00 $1,320.00 TT$4,100.00 $650.79 TT$4,216.00 $669.21Scientific Equipment TT$5,499.90 $873.00Other TT$3,458.70 $549.00

Phase II: Project ImplementationStipend TT$11,340.00 $1,800.00 TT$0.00 $0.00 TT$11,340.00 $1,800.00Local Guides TT$4,725.00 $750.00 TT$0.00 $0.00 TT$4,725.00 $750.00Transportation TT$15,120.00 $2,400.00 TT$20,685.75 $3,283.45 -TT$5,565.75 -$883.45Logistics/ Living Expenses TT$15,246.00 $2,420.00 TT$15,201.40 $2,412.92 TT$44.60 $7.08Education/Awareness TT$3,811.50 $605.00 TT$10,770.99 $1,709.68 -TT$6,959.49 -$1,104.68Laboratory Testing TT$3,015.90 $478.71 -TT$3,015.90 -$478.71

Phase III: Post Project ExpensesAdministration TT$315.00 $50.00 TT$0.00 $0.00 TT$315.00 $50.00Report preparation TT$1,260.00 $200.00 TT$0.00 $0.00 TT$1,260.00 $200.00Contingency TT$6,507.90 $1,033.00 TT$0.00 $0.00 TT$6,507.90 $1,033.00

Total CLP Grant TT$78,750.00 $12,500.00

Additional FundingCI Seed grant (used for lab testing TT$14,882.99 $2,362.38 TT$14,882.99 $2,362.38 TT$0.00 $0.00

Exchange RateTotal TT$93,632.99 $14,862.38 TT$80,380.58 $12,758.82 TT$13,252.41 $2,103.56

Grant Proposal budget Actual Expenditure Difference

6.3

TT$11,723.55 $1,860.88 TT$2,764.95 $438.88

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